The present invention relates to methods of detecting an early stage of renal disease and/or renal complications of a disease. The invention also relates to preventing and treating the disease.
The appearance of excess protein such as albumin in the urine is indicative of kidney disease. Diabetic nephropathy is such a disease. By the time the excess albumin is detected, kidney disease has progressed, possibly to a stage where it is irreversible and treatment has little effect. Therefore it is an object of the invention to provide a test that is more sensitive than the currently known radioimmunoassay to detect such a disease as early as possible so that the disease can be either prevented or a treatment protocol commenced early on in the disease.
Specific proteinuria, and in particular, albuminuria (micro- and macro-), is a marker of disease including renal disease (glomerulonephritis, bacterial and viral glomerulonephritides, IgA nephropathy and Henoch-Schxc3x6nlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjxc3x6gren""s syndrome, diabetic nephropathy, nephrotic syndrome (minimal change disease, focal glomerulosclerosis and related disorders), acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Preclampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis), genetic renal disease (medullary cystic, medullar sponge, polycystic kidney disease (autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tuborous sclerosis), von Hippel-Lindau disease, familial thin-glomerular basement membrane disease, collagen III glomerulopathy, fibronectin glomerulopathy, Alport""s syndrome, Fabry""s disease, Nail-Patella Syndrome, congenital urologic anomalies), monoclonal gammopathies (multiple myeloma, amyloidosis and related disrders), febrile illness (familial Mediterannean fever, HIV infectionxe2x80x94AIDS), inflammatory disease (systemic vasculitides (polyarteritis nodosa, Wegener""s granulomatosis, polyarteritis, necrotizing and crescentic glomerulonephritis), polymyositisdermatomyositis, pancreatitis, rheumatoid arthritis, systemic lupus erythematosus, gout), blood disorders (sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute corticol necrosis, renal thromboembolism), trauma and surgery (extensive injury, bums, abdominal and vascular surgery, induction of anesthesia), drugs (penicillamine, steroids) and drug abuse, malignant disease (epithelial (lung, breast), adenocarcinoma (renal), melanoma, lymphoreticular, multiple myeloma), circulatory disease (myocardial infarction, cardiac failure, peripheral vascular disease, hypertension, coronary heart disease, non-atherosclerotic cardiovascular disease, atherosclerotic cardiovascular disease), skin disease (psoriasis, systemic sclerosis), respiratory disease (COPD, obstructive sleep apnoea, hypoia at high altitude) and endocrine disease (acromegaly, diabetes mellitus, and diabetes insipidus).
Kidney disease may result from bacterial infection, allergies, congenital defects, stones, antibiotics, immunosuppressives, antineoplastics, nonsteroidal anti-inflammatory drugs, analgesics, heavy metals, tumors, chemicals.
The applicant has found that proteins, including albumin, are normally excreted as a mixture of native protein and fragments that are specifically produced during renal passage (Osicka, T. M. et al. (1996) Nephrology, 2, 199-212). Proteins are heavily degraded during renal passage by post-glomerular (basement membrane) cells, which may include tubular cells. Lysosomes in renal tubular cells may be responsible for the breakdown of proteins excreted during renal passage (see FIG. 1). The breakdown products are excreted into the tubular lumen. In normal individuals, most of the albumin in the urine is fragmented.
When lysosome activity or intracellular processes directing substrates to lysosomes is reduced, more of the high molecular weight, and substantially full length albumin appears in the urine. This reflects an imbalance in the cellular processes in the kidney tissue.
Until now, it was thought that conventional radioimmunoassay was suitable for detecting all (total) of a specific protein in a sample. But the total content of the protein may include more than those that are identifiable by known antibodies using conventional radioimmunoassay (RIA). Currently available radioimmunoassays rely on antibodies to detect proteins such as albumin. Antibody detection is very accurate down to nanogram levels. However, the specificity of the antibodies influences detection of the protein. The antibody detects certain epitopes. If the specific epitope on the albumin is absent, altered or masked, or the albumin is modified in any other way so that the antibody fails to detect the albumin, conventional radioimmunoassays may not provide a true representation of the true amount of albumin present in a urine sample.
Methods of detecting early signs of a disease, including kidney disease, determining a patient""s propensity for the disease, preventing the onset of the disease, and treating the disease at the earliest stage possible, as well as a method for determining the total content of a specific protein in a sample, are some of the objects of the invention.
The present invention is directed to a method of diagnosing early stage of renal disease and/or renal complications of a disease, comprising:
(a) separating all of the proteins in a urine sample; and
(b) detecting a modified form of a protein in the sample, wherein detection of the modified protein is indicative of an early stage of the renal disease and/or renal complications of a disease.
Although not limited to any particular disease, according to the method of the invention, the disease sought to be diagnosed includes nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease (glomerulonephritis, bacterial and viral glomerulonephritides, IgA nephropathy and Henoch-Schxc3x6nlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjxc3x6gren""s syndrome, nephrotic syndrome (minimal change disease, focal glomerulosclerosis and related disorders), acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis), genetic renal disease (medullary cystic, medullar sponge, polycystic kidney disease (autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tuborous sclerosis), von Hippel-Lindau disease, familial thin-glomerular basement membrane disease, collagen III glomerulopathy, fibronectin glomerulopathy, Alport""s syndrome, Fabry""s disease, Nail-Patella Syndrome, congenital urologic anomalies), monoclonal gammopathies (multiple myeloma, amyloidosis and related disorders), febrile illness (familial Mediterranean fever, HIV infectionxe2x80x94AIDS), inflammatory disease (systemic vasculitides (polyarteritis nodosa, Wegener""s granulomatosis, polyarteritis, necrotizing and crescentic glomerulonephritis), polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis, systemic lupus erythematosus, gout), blood disorders (sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute cortical necrosis, renal thromboembolism), trauma and surgery (extensive injury, bums, abdominal and vascular surgery, induction of anesthesia), drugs (penicillamine, steroids) and drug abuse, malignant disease (epithelial (lung, breast), adenocarcinoma (renal), melanoma, lymphoreticular, multiple myeloma), circulatory disease (myocardial infarction, cardiac failure, peripheral vascular disease, hypertension, coronary heart disease, non-atherosclerotic cardiovascular disease, atherosclerotic cardiovascular disease), skin disease (psoriasis, systemic sclerosis), respiratory disease (COPD, obstructive sleep apnoea, hypoia at high altitude) and endocrine disease (acromegaly, diabetes mellitus, diabetes insipidus).
In addition, the method can be practiced using any protein, preferably, albumin, globulin (xcex1-globulin(xcex11-globulin, xcex12-globulin),xcex2-globulin,xcex3-globulin), euglobulin, pseudoglobulin I and II, fibrinogen, xcex11 acid glycoprotein (orosomucoid), xcex11 glycoprotein, xcex11 lipoprotein, ceruloplasmin, xcex12 19 S glycoprotein, xcex21 transferrin, xcex21 lipoprotein, immunoglobulins A, E, G, and M, horseradish peroxidase, lactate dehydrogenase, glucose oxidase, myoglobin, lysozyme, protein hormone, growth hormone, insulin, or parathyroid hormone.
The method can be practiced using non-antibody means, using such methods as chromatography, electrophoresis, or sedimentation, which further include such methods as partition chromatography, adsorption chromatography, paper chromatography, thin-layer chromatography, gas-liquid chromatography, gel chromatography, ion-exchange chromatography, affinity chromatography, or hydrophobic interaction chromatography, moving-boundary electrophoresis, zone electrophoresis, or isoelectric focusing. Albumin detection with specific albumin dyes may also be used.
In particular, the method of the invention is directed to using a hydrophobic interaction chromatography in a high-pressure liquid chromatography (HPLC) apparatus.
The present invention is also directed to an antibody detecting method for diagnosing early stage of renal disease and/or renal complications of a disease. The method of the invention is accomplished by assaying for an intact/modified protein that is not normally identifiable in urine using conventional means. The intact/modified protein of the invention is present in the urine sample of a diseased person or a person who is predisposed to a disease before the native protein can be detected. Therefore, the detection of an intact/modified protein in a urine sample indicates at an early stage that the subject is either diseased or predisposed to the disease, even though the subject may otherwise appear to be normal. An assay method of the invention includes detecting an intact/modified protein by an antibody specific for both the modified and unmodified forms of the protein. Preferably, the antibody is specific for the modified protein. The antibody can be attached to an enzymatic, radioactive, fluorescent or chemiluminescent label, wherein the detecting step comprises radioimmunoassay, immunoradiometric assay, fluorescent immunoassay, enzyme linked immunoassay, or protein A immunoassay.
In the method of the invention, the early stage of the disease is diagnosed when the modified protein is present in the urine in increasing amounts over time, and conventional radioimmunoassay does not detect the modified protein.
The present invention is also directed to an article of manufacture for diagnosing an early stage of renal disease and/or renal complications of a disease, comprising:
(a) a container comprising a labeled antibody specific for a modified form of the protein;
(b) a container comprising reagents for developing antibody reaction; and
(c) instructions on how to use components (a) and (b) to carry out the diagnosis.
In addition, the present invention is also directed to a method for determining a treatment agent for renal disease and/or renal complications of a disease, comprising:
(a) administering to a person in need thereof an agent that is suspected of being able to treat the disease;
(b) obtaining a urine sample from the person; and
(c) assaying for a modified form of the protein in the sample, wherein either presence or lack of presence of the modified form of the protein in the urine or decreasing amount of the modified form of the protein over time indicates that the agent is a treatment agent for the renal disease and/or renal complications of a disease.
The invention is also directed to a method for treating a person suffering from a disease in which a diseased state is indicated by specific proteinuria, comprising administering a therapeutically effective amount of the treatment agent obtained according to the above method to a person in need thereof. Preferably, the treatment agent is a lysosome activating compound.
Another object of the invention is to determine the sum of modified and unmodified forms of a of a specific protein in a sample, comprising:
(a) separating all of the proteins in the sample;
(b) detecting modified and unmodified forms of the specific protein; and
(c) integrating the modified and unmodified forms of the specific protein to determine the sum of the specific protein in the sample.
Preferably, the sample is a biological sample, such as urine.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.